BIOL21351 Cell Death L0-2

Pathology of cancer (4)

1. Mutation

2. Tumour growth

3. Intravasion - Spread of malignant cells

4. Metastasis - Tumour growth in more than 1 organ

Mutations in which genes play a significant role on cancer? What are the types of mutations involved?

Activating mutations in oncogenes

Inactivating mutations in tumour repressor genes

First identified oncogenes

Tyrosine Kinase Src

Role of PK in cancer

When it is switched on, it phosphorylates enzymes, leading to proliferation

What is the result of Philadelphia Chromosome translocation?

A translocation between chromosome 9 and 22 leads to a BCR-ABL fusion gene that overexpresses tyrosine kinase (an oncogene) leading to cell proliferation and most commonly chronic myologenous leukemia (CML)

Behaviour of cell in necrosis

Cell swells, PM integrity is lost and organelles are released to the surrounding tissue

Behaviour of cells in apoptosis

Cell shrinks, PM integrity is maintained, apoptotic bodies are phagocytosed by macrophages

Behaviour of cell in autophagy

PM integrity is maintained, organelles are degraded and recycled

Apoptosis is an...

Active process regulated by proteinases

Morphological changes in apoptosis (5)

- Cell shrinks and detaches from neighbor

- Blebs may form on the cell surface

- Chromatin condensation and nuclear fragmentation - ordered process resulting in small membrane bound packets

- Fragmentation of cell into membrane-bound packets (apoptotic bodies)

- Phagocytosis of apoptotic bodies by macrophages - little or no inflammatory reaction.

Proteinases involved in apoptosis

Caspases

What is the site of action of caspases? (I.e. where in the protein do they cleave)

They are endopeptidases that cleave at Asp amino acid, usually leading to activation of function

When caspases are activated, they activate _____ by inhibiting ______

CAD - Caspase Activating DNase

ICAD - Inhibitor of Caspase Activating DNase

The on/off switch of apoptosis is

The activation of caspases

How are initiator caspases expressed when not activated?

As a monomeric proenzyme

List the initiator caspases

1, 2, 8, 9, 10

Difference in the structure of initiator and executioner caspases

Initiator caspases have a large pro-domain whereas executioner is very small

How are initiator caspases activated?

A signal that results in their dimerisation

Difference in how Caspase 8 and 9 activate

Caspase 8 is activated by FasL binding to Fas/CD95 receptor which causes the recruitment of FADD and Caspase 8

Caspase 9 is activated when cytochrome C is released from mitochondria and binds to 7 APAF-1 units to create a cyclic heptamer that loads Caspase-9

Role of initiator caspases in apoptosis

Amplify signal by activating multiple executioner caspases

List the executioner caspases

3, 6, 7

How are executioner caspases expressed when not activated?

Dimer proenzyme

How are executioner caspases activated?

They are cleaved by initiator caspases

Role of executioner caspases

Carry out apoptosis - cause DNA fragmentation, membrane blebbing and form apoptotic bodies

Why do caspases not dimerise spontaneously in the cell?

As the kd is 50μM but the concentration in the cell is 9-20μM so requires the large pro-domain to form the activation complex

Pro-domains of Caspase 1, 8 and 9 and their roles

1 & 9 - CARD

8 - DED/DED

Allows recruitment to the activation complex

Activation of Caspase 9

Cytochrome C (released from mitochondria) binds to APAF-1 to form a cyclic heptamer that binds to Caspase 9 at the CARD domain, causing it to dimerise

What role does the Bcl-2 family play in cell death?

It regulates apoptosis through the mitochondrial outer membrane permeablisation

Problems caused by mutation in cytochrome C

Changes the APAF-1 binding site so blocks apoptosis formation (no affect on respiration)

Function of Bcl-2

It is an anti-apoptosis factor that stops cell death, but not proliferation

Mutation of Bcl-2 and its cause

Reciprocal translocation between t(14:18) —> Bcl-2

18q21 —> Bcl-2 overexpression —> No cell death

How is the mutation in Bcl-2 generated?

By defective VDJ rearrangements during B-cell development

Role of anti-apoptotic factors

They prevent holes being formed in the OMM

List the anti-apoptotic factors

Bcl-2, Bcl-W, Bcl-X, Mcl-1

List the pro-apoptosis factors

Bok, Bax, Bak

List the BH3-only proteins

Bid, Bad, Bim, Bik, PUMA

Role of BH3-only proteins

They inhibit the activity of anti-apoptotic factors and stimulate the activity of pro-apoptotic factors

Which BH3-only proteins are activated by which signals?

Death receptors : Bid

Kinases. : Bad

DNA Damage. : PUMA —> Bax

Growth factors. : Bim

How can the threshold for Bcl-2 regulation of MOMPs be shifted?

By mutations in oncogenes and tumour suppressor genes

What happens when EGF binds to EGFR?

EGFRs dimerise and activate the kinase activity of the receptors (autophosphorylation by P13K) which phosphorylates and activates Akt which phosphorylates Bad, inactivating it by binding it to 14-3-3 protein

Two types of oncogenic mutations in EGFRs

Too many EGFR receptors (cancer)

Too many anti-apoptotic factors (leukemia)

Too many EGFR receptors leads to...

Too much phosphorylation of Bad, so it is never activated so can never stimulate pro-apoptotic factors so no cell death

Too many anti-apoptotic proteins in the EGFR pathway leads to...

Anti-apoptotic factors are more likely to be inhibited by Bad than pro-apoptotic factors are activated so no holes made in OMM —> no cell death

What is p53? Is it common in cancer?

P53 is a tumour suppressor mutated in 50% of cancers

How does a cell respond to DNA damage (involving p53)

DNA damage —> p53 phosphorylated —> activation of PUMA —> activation of Bax —> Cytochrome C released —> Apoptosis

Way to target cancer caused by non-functional apoptosis

Use BH3-only activating drugs —> leads to apoptosis

When do BH3-only activating drugs not work in chemotherapy?

When the p53 gene is mutated

How does BH3-only protein interact with apoptotic factors?

They interact with the BH3 domain (an a-helix) on the apoptotic proteins

How can NMR spectroscopy be used to aid cancer treatments regarding non-functional apoptosis? Give an example

It can be used to identify small molecules that bind to the BH3 domain on anti/pro-apoptotic factors. E.g. two molecules with low affinity were found to do this but when combined together they have greater affinity - ABT-737

Cancer treatments for non-functional apoptosis (2)

ABT-737 and Venetoclax

How does Venetoclax work?

It binds to and inhibits Bcl-2, a useful treatment in Leukemia

How is Caspase-8 activated?

FasL binds to Fas/CD95, which recruits FADD, which recruits Caspase 8 (by DED), forming the DISC, and causes it to dimerise, leading to activation.

What family is CD95/Fas part of?

TNFR (Tumour Necrosis Factor receptor)

Structure of CD95/Fas

It is a trimeric transmembrane domain receptor

What happens once Caspase 8 is activated?

It is cleaved (stabilising the active dimer) and activates executioner caspases

What is in the DISC?

death inducing signalling complex

It involves FADD and Caspase-8 and activates Caspase 3

What activates and what inhibits Caspase 3?

Caspase 8 activates Caspase 3

XIAP inhibits Caspase 3

What can lead to ALPS?

Defects in FasL, CD95 or in Caspase 8

What effect does ALPS have in the cell?

Accumulation of T-lymphocytes, autoimmune conditions & lymphoid tumours

What is ALPS?

Acute lymphoproliferative Syndrome - an autoimmune disease caused by inactivating mutations in FasL, CD95 and Caspase 8

Why is FasL not possible in cancer treatments?

It interacts with many cell types like hepatocytes and so would result in loss of liver function and is toxic

Possible cancer treatment via death receptor ligands

TRAIL receptors DR4 and DR5

Similarities of apoptotic and non-apoptotic caspases

They are activated by the same mechanisms but have different signals and substrates

What leads to Caspase 1 activation initiation?

Damaged/ stressed cells release DAMPs which cause the oligomerisation of NLRs —> formation of inflammosomes

Types of PAMPs

Bacterial cell wall component, microbial DNA

Types of DAMPs

ATP, heat shock proteins and uric acid crystals

Where are PAMP and DAMP signals released from?

PAMPs are released from pathogens and alert immune system

DAMPs are released from damaged / stressed cells

Outline the specific mechanism for Caspase 1 activation

NLR's PYD-ASC adaptor protein (PYD-CARD)-Caspase 1 at CARD domain - Caspase 1 dimerisation

What does Caspase 1 do when activated?

Processes inflammatory cytokines : Pro-IL-B —> IL-B

What causes necrotic cell death?

Toxicity, release of DAMPs/ PAMPs

Morphological characteristics of necrosis

Cell swells, plasma membrane integrity is lost, DAMPs release —> inflammation

The role of ischaemia in cell death

Ischaemia is the loss of blood supply so no ATP is produced and leads to cell death by necrosis

What is reperfusion?

Restoration of blood flow

How can reperfusion injury occur?

Decreased intracellular K+ —> Increased intracellular Ca2+ —> Nox (NADPH oxidase) activation and protease chaplain —> Increased H+ (acidification of the cytosol) —> decreased cellular energy levels by activating enzymes (e.g. PARP)

How do mitochondria respond to decreased pH?

The permeability transition pores are opened

What is the permeability transition pore?

Opening of large pores that leads to mitochondrial cell death

Outline how PTP leads to mitochondrial cell death

PTP opens —> immediate loss of membrane potential —> No ATP generation —> solutes enter the matrix and mitochondria swell and burst

How is the mitochondrial permeability transition distinguished from Mitochondria permeabilisation?

MPT does not require Cytochrome C or ATP - necrosis

MP requires Cytochrome C and ATP - apoptosis

What is secondary necrosis?

Cells that have already gone under apoptosis are further degraded

How does secondary necrosis occur?

No potential —> No ATP production in mitochondria

What is the role of Cyclosporine A?

It inhibits Cyclophilin D, (which is part of the PTP) and plays a role in mitochondria protection and decreases stroke damage by decreasing necrosis

Outline how necrosis induces apoptosis in neighbouring cells

1. Release of ROS (Reactive Oxygen Species)

2. ROS acts as DAMPs, inducing an inflammatory response

3. Death receptor ligands are recruited —> Apoptosis

What is the role of autophagy?

In times of stress, autophagy allows self degradation of mitochondria, proteins, the ER, peroxisomes etc to release needed nutrients or for housekeeping

What does autophagy NOT involve? (4)

Membrane blebbing

Nuclear condensation

DNA fragmentation

Caspase activation

Generally, how does autophagy occur?

Autophagosomes (large vacuoles) fuse with lysosomes and the contents are degraded and recycleed

Autophagy is associated with...

Specific markers that can be seen on the cytoplasmic vacuoles and regulated by signalling complexes

Outline the mechanism by which autophagy can occur

. PIP2 —> PIP3.

ATG13 complex —> Beclin1/VPS34 complex —> ATG5-12 complex —> ATG7/3 which converts LC3 —> LC3-PE

OR

ULK1 —> PI3K complex / Beclin1/VPS34 —> ATG5-12 complex —> ATG3/7

PIP2 —> PIP3

When nutrients are low...

Akt inactive and AMPK active

MTOR is inhibited which allows the ATG13 / ULK1 complex to stimulate autophagy

When there are lots of nutrients...

Akt is active and AMPK is inactive

mTOR is activated so it inhibits the ATG13 and ULK1 complex so autophagy is prevented

What does AMPK do in autophagy?

It activates the ULK1 complex

It inhibits the mTOR complex

What happens to cancer cells if there are low nutrients?

They go into autophagy to prevent the cell from dying by apoptosis

Define mitophagy

A specialised form of autophagy that selectively degradades mitochondria

What causes Parkinson's disease in the brain?

Decreased dopamine neurones in the substantia nigra

How do people get Parkinson's disease? Inherited?

90% sporadic, 10% inherited (autosomal recessive)

What is the role of PINK and Parkin?

They label defective mitochondria with poly-ubiquittin which targets them to the autophagosome for degradation

Outline the steps by which defective mitochondria is targeted to autophagy

Loss of mitohondrial coupling/ membrane potential —> Defective mitochondrial coupling (loss of membrane potential) —> PINK accumulates at mitochondria —> Parkin recruited —> mitochondrial proteins are labelled with Poly-Q and bind to p62 —> targets mitochondria to LC3-labelled autophagosomes

Specifically, which part of initiator caspases allows for their recruitment to the activating complex?

The N-terminal pro-domain

Activating complexes for Caspase 1, 8 & 9

1 - inflammosome

8 - DISC

9 - apoptosome

How is ATP used in the formation of the apoptosome?

(D)ATP binds to NOD, it is hydrolysed to (d)ADP upon Cytochrome C binding and is swapped for a new (d)ATP to form the apoptosome and recruit Caspase 9

How is a threshold for MOMP set?

By Bcl-2 family proteins (anti and pro-apoptotic